Bottom Line:
Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes.Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17.Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function.

ABSTRACTNeural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.

f2: Involvement of ADAM12 and ADAM17 in hypoxia-induced disruption of neural vascular barrier.(a) Immunofluorescence images for claudin-5 expression in CHX-untreated or treated bEnd.3 monolayers in the presence or absence of MG-132. (b) Immunofluorescence images for claudin-5 expression in CHX-untreated or treated bEnd.3 monolayers in the presence or absence of TAPI-1. (c) Quantitative analysis of claudin-5 levels on cell membranes corresponding to the images in a. (d) Quantitative analysis of claudin-5 levels on cell membranes corresponding to the images in b. (e) RT-PCR for the expression of ADAM family members in bEnd.3 cells under normoxia. (f) Immunofluorescence images for claudin-5 of normoxic or hypoxic bEnd.3 monolayers pretreated with the siRNA specific for each member of ADAM family. Two kinds of siRNAs were designed to suppress the expression of each member, and the representative photographs are presented. Hypoxia-induced disappearance of claudin-5 from cell membranes is inhibited with the pretreatment of siRNAs for ADAM12 or ADAM17. (g) TEERs of bEnd.3 monolayers under normoxia or hypoxia after the induction of siRNAs for ADAM12 or ADAM17, showing that the suppression either ADAM12 or ADAM17 rescues bEnd.3 monolayers from the impairment of barrier properties under hypoxia. *P < 0.01; **P < 0.05; ns, not significant. NC siRNA; non-silencing siRNA for negative control.

Mentions:
Under normoxic condition, the presence of MG-132, an inhibitor of ubiquitin-proteasome system, suppressed the disappearance of claudin-5 from cell membranes in CHX-treated cells completely to the level of normoxic cells without CHX treatment (Fig. 2a,c), which is consistent with the results of Mandel I et al. with HeLa cells14. On the other hand, under hypoxic condition, MG-132 treatment rescued CHX-treated hypoxic cells from the disappearance of claudin-5 only partially, not to the level of CHX-untreated normoxic cells (Fig. 2a,c). These data indicate that the physiological turnover of claudin-5 under normoxia is ubiquitin-proteasome system-dependent, while other additional mechanisms must be activated under hypoxia to accelerate the turnover of claudin-5. Based on our previous data showing the post-transcriptional regulation of claudin-5 expression under hypoxia9, we hypothesized that some proteases which could process claudin-5 molecules within 30 minutes after hypoxic stimulation accelerate the hypoxic disappearance of claudin-5. ADAMs were thought to be the candidates for responsible proteases, since they are known to be the modulators for processing of various membrane molecules in a manner of quick response to stimuli151617. As shown in Fig. 2b,d, the addition of TAPI-1, an inhibitor of ADAMs, to the culture medium of cells without CHX treatment abolished the hypoxic loss of claudin-5. It is noteworthy that, in the presence of CHX, TAPI-1 rescued the endothelial cells from hypoxic loss of claudin-5 to the level of CHX-treated normoxic cells but not to the level of CHX-untreated normoxic cells (Fig. 2b,d). These findings indicate that some of ADAM family members accelerate the turnover of claudin-5 under hypoxic, not under normoxic, condition.

f2: Involvement of ADAM12 and ADAM17 in hypoxia-induced disruption of neural vascular barrier.(a) Immunofluorescence images for claudin-5 expression in CHX-untreated or treated bEnd.3 monolayers in the presence or absence of MG-132. (b) Immunofluorescence images for claudin-5 expression in CHX-untreated or treated bEnd.3 monolayers in the presence or absence of TAPI-1. (c) Quantitative analysis of claudin-5 levels on cell membranes corresponding to the images in a. (d) Quantitative analysis of claudin-5 levels on cell membranes corresponding to the images in b. (e) RT-PCR for the expression of ADAM family members in bEnd.3 cells under normoxia. (f) Immunofluorescence images for claudin-5 of normoxic or hypoxic bEnd.3 monolayers pretreated with the siRNA specific for each member of ADAM family. Two kinds of siRNAs were designed to suppress the expression of each member, and the representative photographs are presented. Hypoxia-induced disappearance of claudin-5 from cell membranes is inhibited with the pretreatment of siRNAs for ADAM12 or ADAM17. (g) TEERs of bEnd.3 monolayers under normoxia or hypoxia after the induction of siRNAs for ADAM12 or ADAM17, showing that the suppression either ADAM12 or ADAM17 rescues bEnd.3 monolayers from the impairment of barrier properties under hypoxia. *P < 0.01; **P < 0.05; ns, not significant. NC siRNA; non-silencing siRNA for negative control.

Mentions:
Under normoxic condition, the presence of MG-132, an inhibitor of ubiquitin-proteasome system, suppressed the disappearance of claudin-5 from cell membranes in CHX-treated cells completely to the level of normoxic cells without CHX treatment (Fig. 2a,c), which is consistent with the results of Mandel I et al. with HeLa cells14. On the other hand, under hypoxic condition, MG-132 treatment rescued CHX-treated hypoxic cells from the disappearance of claudin-5 only partially, not to the level of CHX-untreated normoxic cells (Fig. 2a,c). These data indicate that the physiological turnover of claudin-5 under normoxia is ubiquitin-proteasome system-dependent, while other additional mechanisms must be activated under hypoxia to accelerate the turnover of claudin-5. Based on our previous data showing the post-transcriptional regulation of claudin-5 expression under hypoxia9, we hypothesized that some proteases which could process claudin-5 molecules within 30 minutes after hypoxic stimulation accelerate the hypoxic disappearance of claudin-5. ADAMs were thought to be the candidates for responsible proteases, since they are known to be the modulators for processing of various membrane molecules in a manner of quick response to stimuli151617. As shown in Fig. 2b,d, the addition of TAPI-1, an inhibitor of ADAMs, to the culture medium of cells without CHX treatment abolished the hypoxic loss of claudin-5. It is noteworthy that, in the presence of CHX, TAPI-1 rescued the endothelial cells from hypoxic loss of claudin-5 to the level of CHX-treated normoxic cells but not to the level of CHX-untreated normoxic cells (Fig. 2b,d). These findings indicate that some of ADAM family members accelerate the turnover of claudin-5 under hypoxic, not under normoxic, condition.

Bottom Line:
Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes.Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17.Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function.

ABSTRACTNeural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.